Alzheimer's disease (AD) is a neurological disorder that causes the death of neurons in the brain. Generally it progresses slowly, commencing after age 50 years, and its first symptoms may be attributed to old age or to ordinary forgetfulness. As the disease progresses, there is gradual deterioration of cognitive abilities, including the ability to take decisions and carry out everyday tasks, and there may be changes in personality, as well as behavioral problems. In its advanced stages, AD leads to dementia and finally to death. At present the disease is incurable and constitutes a major cause of mortality.
At the present time, Alzheimer's disease is normally diagnosed from the clinical picture, since a definitive diagnosis can only be based on histological examination of brain samples (autopsy or biopsy), revealing the presence of its characteristic features in the brain tissue. In view of the risks associated with brain biopsy in live patients, this procedure is very rarely used, and accordingly it is estimated that the error rate in in-vivo diagnosis of this disease is around 20-30%.
The brains of individuals with Alzheimer's disease show two principal pathological markers: neurofibrillary degeneration (which is identified by the presence of neurofibrillary tangles, dystrophic neurites and neuropil threads) and deposits of amyloid substance (i.e. deposits of so-called β-amyloid peptide, generally abbreviated to Aβ), both in the form of plaques (diffuse plaques and neuritic plaques, these last-mentioned forms being characteristic of the disease, and being designated as such since they appear between and within the neurons), and in the form of vascular deposits (which occur in the walls of the cerebral blood vessels). Both neurofibrillary degeneration and amyloid deposition constitute degenerative processes that are also associated with normal ageing of the brain. In elderly subjects who are not suffering from dementia, the Aβ peptide is mainly deposited in the form of diffuse plaques. This type of deposition is especially pronounced in some subjects with normal cognitive abilities, and for some authors constitutes a process of “pathologic ageing”, which is regarded as being half-way between normal ageing of the brain and AD [1]. Particularly intensive, premature development of diffuse plaques years before neuritic plaques appear also takes place in Down's syndrome (DS), due to trisomy of chromosome 21 which leads to overexpression of the amyloid precursor protein (β-APP). Although a small number of neuritic plaques may be observed in the brains of subjects with normal cognitive abilities, brains affected by AD as well as those affected by DS in the elderly are characterized by the development of a large quantity of mature neuritic plaques [2, 3]. In contrast to the diffuse plaques, which contain a nonfibrilary form of Aβ (designated “preamyloid”) [4], both the vascular amyloid deposits and the neuritic plaques contain Aβ peptide in fibrillary form and react with stains of amyloid substance such as Congo Red and Thioflavine T.
The cognitive decline in AD is correlated linearly with the progression of the neurofibrillary changes and cortical synapse loss [5]. Local synapse loss associated with diffuse plaques has not been observed [6]. In contrast, neuritic plaques are associated with synaptic density loss, neurofibrillary change and activation of the microglia [7]. Both the pure neurofibrillary change and the deposition of Aβ follow well established sequential patterns of progression in AD [8,9]. However, although the degree of cognitive decline has better correlation with a succession of stages based on neurofibrillary change [5], the definitive neuropathologic diagnosis of AD is nevertheless based on histological demonstration of a significantly higher density of neuritic plaques in the associative neocortical regions, relative to that expected according to the patient's age group, within a clinical picture of dementia (consensus criteria of CERAD: Consortium to Establish a Registry for Alzheimer's Disease) [10]. The formation of neuritic plaques constitutes the central pathogenic process in AD, and the molecular composition of these β-amyloid deposits and the differences from the diffuse plaques in relation to said composition is accordingly one of the main fields of interest in current research into AD.
Knowledge about the molecular composition of the β-amyloid deposits in the brain tissue of patients with Alzheimer's disease has changed radically during recent years. Various studies employing biochemical or immunohistochemical methods with the aim of identifying different forms of the Aβ peptide have supplied a fairly consistent picture that permits a molecular interpretation of the classical morphological findings. It was these studies that provided the basis for the unified pathogenic theory of the disease, called the amyloid hypothesis [1,1], although the original hypothesis has been reformulated recently in order to include the emergent role of the soluble Aβ oligomers as the principal pathogenic agents [12]. The assumption of a central and primary pathogenic role of the Aβ peptides in AD is now leading to new therapeutic strategies directed at the prevention or removal of these deposits.
The topographic distribution and temporal sequence of the deposition of Aβ peptides known in brains affected by AD have been elucidated by means of antibodies directed at the carboxyl end, the amino end or to internal segments of the Aβ molecule, together with the isolation and purification of isoforms of Aβ by biochemical methods. The tissue distribution of peptides characterized by the features of their carboxyl ends shows a fairly regular, well-defined pattern. Whereas AβX-40 is the predominant form in the vascular deposits and is the main form encountered in the CSF (cerebrospinal fluid), AβX-42 is the main form detected in the brain tissue deposits (diffuse plaques and neuritic plaques) [13]. In AD, Down's syndrome (DS) [13, 14] and normal ageing [1], AβX-42 is the only component of the diffuse plaques and the principal component of the neuritic plaques. The latter may also contain AβX-40, predominantly in the region of their central nucleus. It has also been established that AβX-42 is the form that is deposited in the initial stages.
Although it was believed initially that AβX-40 and AβX-42 began almost entirely with the amino acid Asp1, it has been well established immunohistochemically and biochemically that a wide variety of heterogeneous isoforms of the Aβ peptide, modified and truncated at the amino end, participate in the composition of both the diffuse and the neuritic plaques [15]. These isoforms also tend to show a regular pattern of distribution between the diffuse and neuritic plaques, so that a complete picture of the topographical distribution of the various Aβ peptides has finally begun to emerge. There are, nevertheless, some discrepancies between the studies, particularly with respect to the characteristics of the amino end of the peptides that make up the diffuse plaques, and the relative amount of amyloid deposit each of them represents.
These discrepancies involve the p3 peptide (Aβ17-42) in particular. Whereas some studies suggest that Aβ17-42 may be the principal component of the diffuse plaques [16], others have found a relatively larger amount of longer forms (beginning at Asp1, or truncated at the amino end or other modified isoforms) at this level [15]. Since Aβ17-42 is produced by excision of β-APP by α-secretase (the so-called nonamyloidogenic pathway) and displays physicochemical properties quite different from the longer isoforms of Aβ (the later being generated by excision of β-APP by γ-secrete), its selective presence in diffuse plaques may be of crucial pathogenic significance in the development of the plaques.
Gowing et al. [17] were the first to isolate the Aβ17-42 peptide as the predominant form recovered from brains affected by AD rich in diffuse plaques. This deposit was not found in vascular amyloid deposits or in neuritic plaques. The commercial monoclonal antibody 6E10, which recognizes Aβ1-17, did not produce immunostaining of diffuse plaques, neither neocortical nor cerebellar, in a series of brains affected by AD and DS [18]. However, the corpus striatum, where the diffuse plaques are particularly abundant in the absence of neuritic plaques, showed some plaques that are positive for the 6E10 antibody. Using HPLC and immunohistochemical determinations, Lalowski et al. [19] demonstrated that Aβ17-42 represents 70% of the total amyloid content in diffuse plaques of the cerebellum, whereas Aβ1-42 represents 12% and other truncated forms of AβX-42 represent 5% or less. In brains of elderly persons affected by DS, Saido et al. [20] found greater staining of diffuse plaques with a specific anti-Aβ N3 (pyroGlu) antibody than with an anti-Aβ N(1) antibody. Iwatsubo et al. [15] studied diffuse plaques in a series of brains from elderly persons, affected by AD and affected by DS with a panel of antibodies directed at recognizing forms of Aβ truncated and modified at the amino end. This study is unique in that the brain tissue employed for the immunohistochemical investigation was fixed either in 70% ethanol, or in 4% formaldehyde, so as to be able to test the effect of routine fixing with formaldehyde on the appearance of artefacts. In all the tissue samples fixed in 70% ethanol, the diffuse plaques were stained intensely by the presence of Aβ N1 (L-Asp), Aβ N1 (L-isoAsp), Aβ N1 (D-Asp), Aβ N3 (pyroGlu), and AβX-42. Weak immunostaining was obtained with Aβ N11 (pyroGlu) and Aβ N17. However, in the material fixed in formaldehyde some diffuse plaques were stained with Aβ N1 (L-Asp), and no staining was obtained with Aβ N1 (L-isoAsp) or Aβ N1 (D-Asp), whereas the pattern of staining for the carboxyl end was unchanged. Although the authors demonstrated that modification of the amino end can alter the results of immunostaining in tissues fixed in formaldehyde, they obtained weak reactivity for Aβ N17 even in material fixed in ethanol. Using a monoclonal antibody specific to p3 (Aβ17-42) [16], they found that deposition of this peptide was largely limited to diffuse plaques, dystrophic neurites and the coronas of neuritic plaques in the regions of the amygdalas, hippocampus and parahippocampus. The authors suggested a specific role of p3 in the initial deposition of amyloid substance and in the origin of the neuritic plaques. Tekirian et al. [21] demonstrated, in a series of brains affected by AD and controls, the presence of Aβ N3 (peptide)>Aβ N1 (D)>Aβ N17 (L)>Aβ N1 (rD) in the diffuse plaques.
With regard to variability at the carboxyl end, in a study using antibodies directed against AβX-40, AβX-42 or AβX-43, Parvathy et al. [22] found a subgroup of neuritic plaques that only react to the Aβ C40 antibody and a larger subgroup of plaques that react both to a Aβ C40 and to Aβ C42.
Earlier studies have thus established that the diffuse plaques show a profile of Aβ that is highly specific at the carboxyl end and a profile that is fairly specific at the amino end, the latter being subject to some heterogeneity between patients and between different regions of the brain. The presence of Aβ17-42 appears to be limited largely to the diffuse plaques, although there is considerable variation between the studies with respect to the relative contents of this shorter peptide in them. In the studies conducted by Kida et al. [18], no staining was obtained for Aβ peptides that included the sequence 12-16 in diffuse plaques of cortical and subcortical regions.
Considered overall, the results of the State of the Art (SA) suggest that, as proposed by Larner [23], the forms of the Aβ peptide that are specific according to the amino end can play an essential role in the development of the diffuse plaques so that they are converted to neuritic plaques. Therefore antibodies capable of specifically detecting forms with the amino end truncated and, accordingly, clearly differentiating between diffuse plaques and neuritic plaques are extremely useful as tools in the diagnosis of Alzheimer's disease. Among them, those that are able to differentiate subgroups of neuritic plaques according to the proportion of the different forms of amyloid peptide that differ in the amino acid terminating the carboxyl end would be especially useful, especially if they could be used for defining subsets of plaques that constitute a specific disease marker. The monoclonal antibody of the invention, directed against the sequence constituted of amino acids 12-16 of β-amyloid peptide and with greater affinity for the Aβ42 form than the Aβ40 form, fulfils both characteristics.
Another aspect in which the antibodies that are able to detect forms of β-amyloid peptide would be particularly useful would be in the diagnosis of Alzheimer's disease by analysis of the concentration of different forms of this peptide in biological fluids, an aspect that is being investigated both to facilitate diagnosis based on biochemical parameters, and even with a view to identifying preclinical cases or those at particular risk of developing the disease and for monitoring patients enrolled in clinical studies. For this purpose, many studies focus on the cerebrospinal fluid (CSF), with the aim of detecting whether there are variations in the concentrations of soluble forms of β-amyloid peptide present in this fluid that make it possible to differentiate between patients and healthy controls. However, it is rather unlikely that analysis of the CSF, which involves the use of an invasive technique, lumbar puncture, could be applied for the diagnosis and routine monitoring of patients with Alzheimer's disease in clinical practice. Therefore other lines of research have focused on investigation of variations in the concentration in blood and urine of biological markers (which include soluble forms of β-amyloid peptide) which might be correlated with the appearance and development of the disease, although few studies have been published to date. According to these, the plasma levels of AβX-42 and AβX-40 appear to be increased if there is Down's syndrome and they also increase in normal individuals with age. The plasma levels of AβX-42, but not of AβX-40, are raised in patients with Alzheimer's disease, and decrease as the disease develops [28, 29], although measurement of the concentration of these species in plasma cannot currently be applied in diagnosis. The presence of soluble forms of β-amyloid peptide has also been detected in urine [30], although a comparative study between patients and controls has not yet been carried out. Although the order of magnitude of the concentrations of β-amyloid peptide that are being detected in plasma and urine are making it difficult to define the profiles corresponding to healthy individuals and to patients in different stages of the disease and the possible association of the soluble forms of β-amyloid peptide with other proteins present in said biological fluids is another difficulty to be overcome, this is an approach being actively pursued, so that the monoclonal antibodies that can interact with forms of β-amyloid peptide present in biological fluids to make it possible to detect them can represent a very useful tool in the diagnosis of Alzheimer's disease. The antibody of the invention, which is able to interact with soluble forms of β-amyloid peptide and detect their presence in biological fluids such as urine, is one of the antibodies that may be useful for implementing these diagnostic techniques. Moreover, the fact that it is directed specifically against the region close to the amino end of β-amyloid peptide is a characteristic of interest in the differentiation of the forms of β-amyloid peptide that conserve the amino end from those forms in which said end is truncated.
Other monoclonal antibodies directed specifically against the region close to the amino end of β-amyloid peptide, as well as their use in diagnostic methods connected with AD, have been described. For example, U.S. Pat. No. 4,666,829 describes the production of a monoclonal antibody generated against a portion of the amyloid peptide closest to the amino end. Concretely, a synthetic peptide was prepared comprising the first ten residues of said peptide (Asp-Ala-Glu-Phe-Arg-His-Asp-Ser-Gly-Tyr), represented by SEQ ID NO: 1. The epitope recognized by this antibody % kill be included in these amino acids 1 to 10, whereas the antibody claimed in the present invention recognizes at least the epitope that comprises the amino acids from 12 to 16. As well as differing in the specific sequence that recognizes this monoclonal antibody with respect to that of the present invention, the design of the proposed diagnostic technique is also different, since what is determined is exclusively the binding of the antibodies used to what is considered in this patent to be the peptide that is characteristic of Alzheimer's disease, that constituted by amino acids 1 to 28 of the amyloid peptide, without considering binding to other forms of the peptide of different lengths and/or with variations in the amino and carboxyl ends. Moreover, no experimental proof is given that demonstrates its capacity for detecting forms of the amyloid peptide present in biological fluids.
Furthermore, patent application PCT WO 90/12871 describes the preparation of the monoclonal antibody designated SV17-6E10. This antibody is generated by immunization with the peptide sequence Asp-Ala-Glu-Phe-Arg-His-Asp-Ser-Gly-Tyr-Gln-Val-His-His-Gln-Lys-Leu, represented by SEQ ID NO:2, which might be considered equivalent to that of amino acids 1 to 17 of the amyloid peptide. Another monoclonal antibody directed against the region that includes amino acids 1 to 17 is the aforementioned commercial monoclonal antibody 6E10 which, as pointed out in the description of the data sheet corresponding to the product, http://www.alexis_corp.com/monoclonal:antibodies-SIG-9320/opfa.1.1.SIG-9320.386.4.1.html, specifically, within amino acids 1 to 17 of β-amyloid peptide, recognizes the epitope comprised between amino acids 3 to 8. This epitope corresponds to a different region, closer to the amino end than that of the EM5 monoclonal antibody of the invention. Although it is capable of producing differential staining of neuritic plaques and vascular deposits, without staining the diffuse plaques, this antibody does not appear to display differences in affinity between peptides Aβ42 and Aβ40.
Accordingly, the monoclonal antibody of the invention, which has been designated EM5, constitutes a novel tool, because it recognizes at least one epitope that is not recognized by any of the antibodies described in the known state of the art, in the sequence of β-amyloid peptide. The antibody of the invention is, accordingly, useful for application in the diagnosis of Alzheimer's disease. It detects neuritic plaques specifically without detecting diffuse plaques, which are not specifically associated with the disease. Among the neuritic plaques, the monoclonal antibody of the invention makes it possible to detect a subgroup of neuritic plaques differing in their molecular composition relative to the deposits of β-amyloid peptide. Moreover, the monoclonal antibody of the invention is capable of binding to forms of β-amyloid peptide present in solution, permitting subsequent detection and quantification of said peptides, including if the solution is a biological fluid such as urine.